1. Field of the Invention
The present invention relates to a method for the post-mortem detection and localization of degenerating neurons in brain slices, and more particularly to anionic fluorescent type homologue therefor.
2. Prior Art
Many neuroscientists including toxicologists, pathologists, pharmacologists, anatomists and histochemists share an interest in detecting neuronal degeneration. The demonstration of this phenomenon, however, is often difficult from both a technical and an interpretational point of view. Conventional techniques such as H&E (hematoxylin and eosin), or Nissl type stains (crystal violet, thionin, etc.) are technically simple and can be used to infer degeneration based on changes such as neuronal shrinkage, vacuolation, and hyperchromatism. Chassan, J. L., "Nervous System" in W. A. D. Anderson and J. M. Kissane (Eds.), Pathology, Mosby, St. Louis, 1977, pp. 2074-2148; Garcia, J. H. and Kamijiyo, Y., "Cerebral Infraction: Evolution of Histopathological Changes After Occlusion of a Middle Cerebral Artery in Primates," J. Neuropathol. Exp. Neurol., 33 (1974) pp. 409-421; and Siesjo, B. K., "Cell Damage in the Brain: a Speculative Synthesis," Cereb. Blood Metabol., 1 (1981) 155-185. Unfortunately, such changes are not necessarily indicative of neuronal degeneration and may be due to processing artifacts or non-lethal alterations in cellular morphology. Processing artifacts can result in both shrunken and hyperchromatic cells. Cammermeyer. J., "The Importance of Avoiding the "dark" Neurons in Experimental Neuropathology," Acta. Neuropath. 1 (1961) 245-270; and Stensaas, S. S., Edwards, C. Q., and Stensaas, L. J., "An Experimental Study of Hyperchromic Nerve Cells in the Cerebral Cortex," Exp. Neurol., 36 (1972) 472-487. Not only are such techniques prone to false positives, but it is also possible to miss degenerating neurons since all cells stain with these dyes while only relatively subtle morphological differences exist between normal and degenerating neurons. These interpretational difficulties make the analysis of conventionally stained material time consuming. In contrast, suppressed silver techniques are much better from this point of view in that normal neurons remain unstained while degenerating neurons stain black. de Olmos, J. S., Beltramino, C. A., and de Olmos de Lorenzo, S. "Use of an amino-cupric-silver technique for the detection of early and simiacute neuronal degeneration caused by neurotoxicants, hypoxia, and physical trauma," Neurotox.& Teratol., 16, (1994) 545-561; Fink, R. P., Heimer, L., "Two methods for the selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system," Brain Res., 4, (1967) 369-374; Gallyas, F., Wolf J. R., Bottcher, H., and Zaborsky, L., "A reliable and sensitive method to localize terminal degeneration and lysosomes in the central nervous system," Stain Technol., 55 (1980) 299-306; and Nauta, W. J. H. and Gygax, P. A., "Silver impregnation of degenerating axons in the central nervous system: A modified technique," Stain Technol., 29 (1954) 91-93. The main drawbacks associated with suppressed silver techniques lie in their labor intensive and capricious nature.